Exploring the determinants of influenza A/H7N9 control intervention efficacy in China: Disentangling the effect of the '1110' policy and poultry vaccination.

The influenza A virus of the H7N9 subtype (FLUAV H7N9) emerged in Eastern China provinces in 2013 causing illness in both poultry and humans. Most reported FLUAV H7N9 human cases were related to those associated with the live poultry market chain. From 2013 to 2017, there were five epidemic waves of human infections, and from the end of 2016, the number of human cases increased sharply. To control FLUAV H7N9 in the market chain, the so-called '1110' policy at live poultry markets and a national vaccination programme were implemented. The relative efficacy of these two measures on the number of poultry and human infections has not been quantified and compared. To explore their efficacy, a cross-sectional study was conducted in six provinces of China, and the vaccination and surveillance data of H7N9 were analysed. Our survey data showed that poultry vendors were not widely aware of and did not accept the '1110' policy. For subjective and objective factors, some measures of the '1110' policy were not implemented in live bird markets (LBMs). However, the national vaccination programme achieved good immune effects and sharply decreased poultry FLUAV H7N9 infections. The detection rates of FLUAV H7N9 in LBMs and farms gradually decreased since the vaccination programme was implemented. Our analysis also indicated that human infections were closely related to poultry virus carriage rates; therefore, controlling FLUAV H7N9 circulation in poultry was an effective measure to control FLUAV H7N9 infections in humans. Although LBMs play a significant role in human infections, the management measures may not be implemented efficiently; hence, we need to conduct more investigations before developing related policies.

Identification and phylogenetic analysis of five highly pathogenic avian influenza (H5N8) viruses isolated in Urumqi in 2016 / 中华微生物学和免疫学杂志

Objective:To analyze the genetic evolution and molecular characteristics of H5N8 avian influenza viruses (AIVs) isolated from the poultry in a live poultry market (LPM) in Urumqi, Xinjiang.Methods:Oropharyngeal and cloacal swabs of poultry were collected from a LPM in Urumqi in 2016. AIVs were isolated by inoculating swab samples into chicken embryos. Hemagglutination test and RT-PCR were used to identify the AIVs. The genes of isolated AIVs were amplified with the universal primers of AIV and whole-genome sequencing was also performed. Pairwise sequence alignment and analysis of phylogenetic and molecular characteristics were performed using BLAST, Clustal W, MEGA-X and DNAStar software.Results:Five H5N8 AIVs were isolated from poultry. These strains shared a nucleotide identity of 99.70%-100.00%, which indicated that they were from the same source, and were named XJ-H5N8/2016. Phylogenetic analysis based on hemagglutinin( HA), NS and PB2 genes showed that these isolates were clustered together with H5N8 AIVs isolated from the migratory swans in Hubei, Shanxi and Sanmenxia, and the ducks in India during 2016 to 2017. Moreover, they were also clustered together with H5N6 AIVs isolated from minks in China and the first case of human infection in Fujian. The phylogenetic tree of neuraminidase( NA) gene indicated the five isolates clustered together with H5N8 AIVs isolated from ducks in India in 2016, and the phylogenetic trees of PB1, MP, PA and NP genes showed that they were clustered together with H5N8 AIVs isolated from wild birds and poultry in Egypt, Cameroon, Uganda, Congo and other African countries in 2017. The HA cleavage sites of XJ-H5N8/2016 contained five consecutive basic amino acids, indicating high pathogenicity. Multiple mutations in the genes of XJ-H5N8/2016 could enhance its virulence and pathogenicity to mammals. Conclusions:The five strains of H5N8 AIVs isolated from the LPM were highly pathogenic and closely related to the H5N8 AIVs isolated from migratory birds and poultry in Hubei, Shanxi, Sanmenxia area, Africa and India during 2016 to 2017. Meanwhile, some of the viral genes were also closely related to the H5N6 AIVs isolated from the minks and human in China. Multiple mutations could increase the virulence and pathogenicity of AIVs to mammals, which could pose a potential threat to public health.

Comparative evaluation of the transmissibility of SARS-CoV-2 variants of concern

Since the start of the SARS-CoV-2 pandemic in late 2019, several variants of concern (VOC) have been reported, such as B.1.1.7, B.1.351, P.1, and B.1.617.2. The exact reproduction number Rt for these VOCs is important to determine appropriate control measures. Here, we estimated the transmissibility for VOCs and lineages of SAR-CoV-2 based on genomic data and Bayesian inference under an epidemiological model to infer the reproduction number (Rt). We analyzed data for multiple VOCs from the same time period and countries, in order to compare their transmissibility while controlling for geographical and temporal factors. The lineage B had a significantly higher transmissibility than lineage A, and contributed to the global pandemic to a large extent. In addition, all VOCs had increased transmissibility when compared with other lineages in each country, indicating they are harder to control and present a high risk to public health. All countries should formulate specific prevention and control policies for these VOCs when they are detected to curve their potential for large-scale spread.

Integrating PCR-free amplification and synergistic sensing for ultrasensitive and rapid CRISPR/Cas12a-based SARS-CoV-2 antigen detection

Antigen detection provides particularly valuable information for medical diagnoses; however, the current detection methods are less sensitive and accurate than nucleic acid analysis. The combination of CRISPR/Cas12a and aptamers provides a new detection paradigm, but sensitive sensing and stable amplification in antigen detection remain challenging. Here, we present a PCR-free multiple trigger dsDNA tandem-based signal amplification strategy and a de novo designed dual aptamer synergistic sensing strategy. Integration of these two strategies endowed the CRISPR/Cas12a and aptamer-based method with ultra-sensitive, fast, and stable antigen detection. In a demonstration of this method, the limit of detection was at the single virus level (0.17 fM, approximately two copies/L) in SARS-CoV-2 antigen nucleocapsid protein analysis of saliva or serum samples. The entire procedure required only 20 minutes. Given our systems simplicity and modular setup, we believe that it could be adapted reasonably easily for general applications in CRISPR/Cas12a-aptamer-based detection.

A tandem-repeat dimeric RBD protein-based COVID-19 vaccine ZF2001 protects mice and nonhuman primates

A safe, efficacious and deployable vaccine is urgently needed to control COVID-19 pandemic. We report here the preclinical development of a COVID-19 vaccine candidate, ZF2001, which contains tandem-repeat dimeric receptor-binding domain (RBD) protein with alum-based adjuvant. We assessed vaccine immunogenicity and efficacy in both mice and non-human primates (NHPs). ZF2001 induced high levels of RBD-binding and SARS-CoV-2 neutralizing antibody in both mice and NHPs, and also elicited balanced TH1/TH2 cellular responses in NHPs. Two doses of ZF2001 protected Ad-hACE2-transduced mice against SARS-CoV-2 infection, as detected by reduced viral RNA and relieved lung injuries. In NHPs, vaccination of either 25 g or 50 g ZF2001 prevented infection with SARS-CoV-2 in lung, trachea and bronchi, with milder lung lesions. No evidence of disease enhancement is observed in both models. ZF2001 is being evaluated in the ongoing international multi-center Phase 3 trials (NCT04646590) and has been approved for emergency use in Uzbekistan.

Identification of novel bat coronaviruses sheds light on the evolutionary origins of SARS-CoV-2 and related viruses

Although a variety of SARS-CoV-2 related coronaviruses have been identified, the evolutionary origins of this virus remain elusive. We describe a meta-transcriptomic study of 411 samples collected from 23 bat species in a small (~1100 hectare) region in Yunnan province, China, from May 2019 to November 2020. We identified coronavirus contigs in 40 of 100 sequencing libraries, including seven representing SARS-CoV-2-like contigs. From these data we obtained 24 full-length coronavirus genomes, including four novel SARS-CoV-2 related and three SARS-CoV related genomes. Of these viruses, RpYN06 exhibited 94.5% sequence identity to SARS-CoV-2 across the whole genome and was the closest relative of SARS-CoV-2 in the ORF1ab, ORF7a, ORF8, N, and ORF10 genes. The other three SARS-CoV-2 related coronaviruses were nearly identical in sequence and clustered closely with a virus previously identified in pangolins from Guangxi, China, although with a genetically distinct spike gene sequence. We also identified 17 alphacoronavirus genomes, including those closely related to swine acute diarrhea syndrome virus and porcine epidemic diarrhea virus. Ecological modeling predicted the co-existence of up to 23 Rhinolophus bat species in Southeast Asia and southern China, with the largest contiguous hotspots extending from South Lao and Vietnam to southern China. Our study highlights both the remarkable diversity of bat viruses at the local scale and that relatives of SARS-CoV-2 and SARS-CoV circulate in wildlife species in a broad geographic region of Southeast Asia and southern China. These data will help guide surveillance efforts to determine the origins of SARS-CoV-2 and other pathogenic coronaviruses.

A recombinant receptor-binding domain in trimeric form generates completely protective immunity against SARS-CoV-2 infection in nonhuman primates

Safe and effective vaccination is critical to combatting the COVID-19 pandemic. Here, we developed a trimeric SARS-CoV-2 receptor-binding domain (RBD) subunit vaccine candidate that simulates the natural structure of the spike (S) trimer glycoprotein. Immunization with RBD-trimer induced robust humoral and cellular immune responses and a high level of neutralizing antibodies that were maintained for at least 4 months. Moreover, the antibodies that were produced in response to the vaccine effectively neutralized the SARS-CoV-2 501Y.V2 variant. Of note, when the titers of the antibodies dropped to a sufficiently low level, only one boost quickly activated the anamnestic immune response, resulting in complete protection against the SARS-CoV-2 challenge in rhesus macaques without typical histopathological changes or viral replication in the lungs and other respiratory tissues. Our results indicated that immunization with SARS-CoV-2 RBD-trimer could raise long-term and broad immunity protection in nonhuman primates, thereby offering an optimal vaccination strategy against COVID-19.

Recombinant chimpanzee adenovirus AdC7 expressing dimeric tandem-repeat RBD of SARS-CoV-2 spike protein protects mice against COVID-19

A safe and effective vaccine is urgently needed to control the unprecedented COVID-19 pandemic. Four adenovirus vectored vaccines expressing spike (S) protein have advanced into phase 3 trials, with three approved for use. Here, we generated several recombinant chimpanzee adenovirus (AdC7) vaccines expressing S, receptor-binding domain (RBD) or dimeric tandem-repeat RBD (RBD-tr2). We found vaccination via either intramuscular or intranasal route was highly immunogenic in mice to elicit both humoral and cellular (Th1-based) immune responses. AdC7-RBD-tr2 showed higher antibody responses compared with both AdC7-S and AdC7-RBD. Intranasal administration of AdC7-RBD-tr2 additionally induced mucosal immunity with neutralizing activity in bronchoalveolar lavage fluid. Either single-dose or two-dose mucosal administration of AdC7-RBD-tr2 protected mice against SARS-CoV-2 challenge, with undetectable subgenomic RNA in lung and relieved lung injury. These results support AdC7-RBD-tr2 as a promising COVID-19 vaccine candidate.

Neurovirulence of avian influenza virus is dependent on the interaction of viral NP protein with host factor FMRP in the murine brain.

Avian influenza viruses (AIVs) are zoonotic viruses that exhibit a range infectivity and severity in the human host. Severe human cases of AIVs infection are often accompanied by neurological symptoms, however, the factors involved in the infection of the central nervous system (CNS) are not well known. In this study, we discovered that avian-like sialic acid (SA)-α2, 3 Gal receptor is highly presented in mammalian (human and mouse) brains. In the generation of a mouse-adapted neurotropic H9N2 AIV (SD16-MA virus) in BALB/c mice, we identified key adaptive mutations in its hemagglutinin (HA) and polymerase basic protein 2 (PB2) genes that conferred viral replication ability in mice brain. The SD16-MA virus showed binding affinity for avian-like SA-α2, 3 Gal receptor, enhanced viral RNP polymerase activity, increased viral protein production and transport that culminated in elevated progeny virus production and severe pathogenicity. We further established that host Fragile X Mental Retardation Protein (FMRP), a highly expressed protein in the brain that physically associated with viral nucleocapsid protein (NP) to facilitate RNP assembly and export, was an essential host factor for the neuronal replication of neurotropic AIVs (H9N2, H5N1 and H10N7 viruses). Our study identified a mechanistic process for AIVs to acquire neurovirulence in mice.IMPORTANCE Infection of the CNS is a serious complication of human cases of AIVs infection. The viral and host factors associated with neurovirulence of AIVs infection are not well understood. We identified and functionally characterized specific changes in the viral HA and PB2 genes of a mouse-adapted neurotropic avian H9N2 virus responsible for enhanced virus replication in neuronal cells and pathogenicity in mice. Importantly, we showed that host FMRP was a crucial host factor that was necessary for neurotropic AIVs (H9N2, H5N1 and H10N7 viruses) to replicate in neuronal cells. Our findings have provided insights into the pathogenesis of neurovirulence of AIV infection.

Novel reassortant 2.3.4.4B H5N6 highly pathogenic avian influenza viruses circulating among wild, domestic birds in Xinjiang, Northwest China

Background@#The H5 avian influenza viruses (AIVs) of clade 2.3.4.4 circulate in wild and domestic birds worldwide. In 2017, nine strains of H5N6 AIVs were isolated from aquatic poultry in Xinjiang, Northwest China. @*Objectives@#This study aimed to analyze the origin, reassortment, and mutations of the AIV isolates. @*Methods@#AIVs were isolated from oropharyngeal and cloacal swabs of poultry. Identification was accomplished by inoculating isolates into embryonated chicken eggs and performing hemagglutination tests and reverse transcription polymerase chain reaction (RT-PCR). The viral genomes were amplified with RT-PCR and then sequenced. The sequence alignment, phylogenetic, and molecular characteristic analyses were performed by using bioinformatic software. @*Results@#Nine isolates originated from the same ancestor. The viral HA gene belonged to clade 2.3.4.4B, while the NA gene had a close phylogenetic relationship with the 2.3.4.4C H5N6 highly pathogenic avian influenza viruses (HPAIVs) isolated from shoveler ducks in Ningxia in 2015. The NP gene was grouped into an independent subcluster within the 2.3.4.4B H5N8 AIVs, and the remaining six genes all had close phylogenetic relationships with the 2.3.4.4B H5N8 HPAIVs isolated from the wild birds in China, Egypt, Uganda, Cameroon, and India in 2016–2017, Multiple basic amino acid residues associated with HPAIVs were located adjacent to the cleavage site of the HA protein. The nine isolates comprised reassortant 2.3.4.4B HPAIVs originating from 2.3.4.4B H5N8 and 2.3.4.4C H5N6 viruses in wild birds. @*Conclusions@#These results suggest that the Northern Tianshan Mountain wetlands in Xinjiang may have a key role in AIVs disseminating from Central China to the Eurasian continent and East African.

Novel reassortant 2.3.4.4B H5N6 highly pathogenic avian influenza viruses circulating among wild, domestic birds in Xinjiang, Northwest China

Background@#The H5 avian influenza viruses (AIVs) of clade 2.3.4.4 circulate in wild and domestic birds worldwide. In 2017, nine strains of H5N6 AIVs were isolated from aquatic poultry in Xinjiang, Northwest China. @*Objectives@#This study aimed to analyze the origin, reassortment, and mutations of the AIV isolates. @*Methods@#AIVs were isolated from oropharyngeal and cloacal swabs of poultry. Identification was accomplished by inoculating isolates into embryonated chicken eggs and performing hemagglutination tests and reverse transcription polymerase chain reaction (RT-PCR). The viral genomes were amplified with RT-PCR and then sequenced. The sequence alignment, phylogenetic, and molecular characteristic analyses were performed by using bioinformatic software. @*Results@#Nine isolates originated from the same ancestor. The viral HA gene belonged to clade 2.3.4.4B, while the NA gene had a close phylogenetic relationship with the 2.3.4.4C H5N6 highly pathogenic avian influenza viruses (HPAIVs) isolated from shoveler ducks in Ningxia in 2015. The NP gene was grouped into an independent subcluster within the 2.3.4.4B H5N8 AIVs, and the remaining six genes all had close phylogenetic relationships with the 2.3.4.4B H5N8 HPAIVs isolated from the wild birds in China, Egypt, Uganda, Cameroon, and India in 2016–2017, Multiple basic amino acid residues associated with HPAIVs were located adjacent to the cleavage site of the HA protein. The nine isolates comprised reassortant 2.3.4.4B HPAIVs originating from 2.3.4.4B H5N8 and 2.3.4.4C H5N6 viruses in wild birds. @*Conclusions@#These results suggest that the Northern Tianshan Mountain wetlands in Xinjiang may have a key role in AIVs disseminating from Central China to the Eurasian continent and East African.

Isolation, identification and phylogenetic analysis of six strains of H5N6 highly pathogenic avian influenza virus / 中华微生物学和免疫学杂志

Objective:To analyze phylogenetic structure and molecular characteristics of H5N6 avian influenza virus (AIVs) isolated from live poultry market (LPM).Methods:Oropharyngeal and cloacal swabs from poultry, and environmental samples were collected from LPM in Urumqi in December 2018, AIVs were isolated and identified by inoculation of chicken embryo, hemagglutination test and RT-PCR, the viral whole genome was amplified with the universal primers of influenza A virus, and then sequenced, pairwise sequence alignments, phylogenetic and molecular characteristics analysis were performed by BLAST, Clustal W, MEGA-X and DNAStar software.Results:Six strains of H5N6 AIVs were isolated from poultry samples, the identity between the viral genes was high (99.4%-100.0%), so the isolates were the same source. BLAST analysis revealed that the viral NP sequence had the highest identity (99.7%) with H5N6 AIVs isolated from poultry in Suzhou, while the sequence of the remaining 7 viral genes had the highest identity (99.0%-100.0%) with H5N6 AIVs isolated from environment in Guangdong during 2017 to 2018. Phylogenetic analysis showed that the viral HA belonged to Clade 2.3.4.4C, and the viral HA, NA, PB1, PA, NP, and MP were all clustered together with H5N6 AIVs isolated from mink in Eastern China in 2018, while the PB2 and NS were clustered together with H5N6 AIVs isolated from environment in Guangdong from 2017 to 2018. The HA cleavage site contained multiple basic amino acid residues, which was highly pathogenic AIVs (HPAIVs). S137A and T160A mutations of HA could increase binding to human-type receptor SAα2, 6-Gal. Additionally, the viral multiple mutations, including 59-69 deletion in NA, the L89V, G309D, R477G, I495V, I504V, D391E, and A661E in PB2, as well as the P42S, D92E, and 80-84 deletion in NS1, could enhance the viral virulence and pathogenicity to mammals. Conclusions:The 6 strains of H5N6 HPAIVs isolated from LPM have relatively close genetic relationship with H5N6 AIVs isolated from mink in Eastern China and environment in Guangdong during 2017 to 2018, the viral multiple mutations could increase its pathogenicity to mammals, which could pose a potential risk to public health.

Avian influenza A (H7N9) virus: from low pathogenic to highly pathogenic / 医学前沿

The avian influenza A (H7N9) virus is a zoonotic virus that is closely associated with live poultry markets. It has caused infections in humans in China since 2013. Five waves of the H7N9 influenza epidemic occurred in China between March 2013 and September 2017. H7N9 with low-pathogenicity dominated in the first four waves, whereas highly pathogenic H7N9 influenza emerged in poultry and spread to humans during the fifth wave, causing wide concern. Specialists and officials from China and other countries responded quickly, controlled the epidemic well thus far, and characterized the virus by using new technologies and surveillance tools that were made possible by their preparedness efforts. Here, we review the characteristics of the H7N9 viruses that were identified while controlling the spread of the disease. It was summarized and discussed from the perspectives of molecular epidemiology, clinical features, virulence and pathogenesis, receptor binding, T-cell responses, monoclonal antibody development, vaccine development, and disease burden. These data provide tools for minimizing the future threat of H7N9 and other emerging and re-emerging viruses, such as SARS-CoV-2.

Potent synthetic nanobodies against SARS-CoV-2 and molecular basis for neutralization

SARS-CoV-2, the causative agent of COVID-191, recognizes host cells by attaching its receptor-binding domain (RBD) to the host receptor ACE22-7. Neutralizing antibodies that block RBD-ACE2 interaction have been a major focus for therapeutic development8-18. Llama-derived single-domain antibodies (nanobodies, [~]15 kDa) offer advantages including ease of production and possibility for direct delivery to the lungs by nebulization19, which are attractive features for bio-drugs against the global respiratory disease. Here, we generated 99 synthetic nanobodies (sybodies) by in vitro selection using three libraries. The best sybody, MR3 bound to RBD with high affinity (KD = 1.0 nM) and showed high neutralization activity against SARS-CoV-2 pseudoviruses (IC50 = 0.40 g mL-1). Structural, biochemical, and biological characterization of sybodies suggest a common neutralizing mechanism, in which the RBD-ACE2 interaction is competitively inhibited by sybodies. Various forms of sybodies with improved potency were generated by structure-based design, biparatopic construction, and divalent engineering. Among these, a divalent MR3 conjugated with the albumin-binding domain for prolonged half-life displayed highest potency (IC50 = 12 ng mL-1) and protected mice from live SARS-CoV-2 challenge. Our results pave the way to the development of therapeutic nanobodies against COVID-19 and present a strategy for rapid responses for future outbreaks.

A non-competing pair of human neutralizing antibodies block COVID-19 virus binding to its receptor ACE2

Neutralizing antibodies could be antivirals against COVID-19 pandemics. Here, we report the isolation of four human-origin monoclonal antibodies from a convalescent patient in China. All of these isolated antibodies display neutralization abilities in vitro. Two of them (B38 and H4) block the binding between RBD and vial cellular receptor ACE2. Further competition assay indicates that B38 and H4 recognize different epitopes on the RBD, which is ideal for a virus-targeting mAb-pair to avoid immune escape in the future clinical applications. Moreover, therapeutic study on the mouse model validated that these two antibodies can reduce virus titers in the infected mouse lungs. Structure of RBD-B38 complex revealed that most residues on the epitope are overlapped with the RBD-ACE2 binding interface, which explained the blocking efficacy and neutralizing capacity. Our results highlight the promise of antibody-based therapeutics and provide the structural basis of rational vaccine design. One Sentence SummaryA pair of human neutralizing monoclonal antibodies against COVID-19 compete cellular receptor binding but with different epitopes, and with post-exposure viral load reduction activity.

Virus shedding patterns in nasopharyngeal and fecal specimens of COVID-19 patients

Diagnosis is the key point for confirmation and treatment of COVID-19. we focused on comparative analysis of virus dynamics between the upper respiratory and feces specimens in the COVID-19 patients. A total of 66 upper respiratory swabs, 51 feces, 56 urine and 56 plasma samples were sequentially collected from 23 patients in a designated hospital. The plasma and urine samples were all negative, except for urine samples from two severe cases at the latest available detection point. Conversely, virus was shed in respiratory swabs and feces samples during the diseased period. Ten of 12 (83.3%) cases were positive for feces samples, while 14 of 21 (66.7%) were positive for respiratory samples. In addition, the median duration of virus shedding was 10.0 days (IQR 8.0 to 17.0) in the upper respiratory swabs, but was 22.0 days (IQR 15.5 to 23.5) for the feces. Notably, at 26 days after discharge, case 3 (a 45-year-old) was detected positive again in the feces samples, but appears to be healthy and negative for respiratory swabs. These results indicated that beside respiratory samples, intestinal samples (e.g. feces) should be recommended for diagnosis of COVID-19, especially before a patient discharge and for monitoring the relapse of discharged patients.

A novel bat coronavirus reveals natural insertions at the S1/S2 cleavage site of the Spike protein and a possible recombinant origin of HCoV-19

The unprecedented epidemic of pneumonia caused by a novel coronavirus, HCoV-19, in China and beyond has caused public health concern at a global scale. Although bats are regarded as the most likely natural hosts for HCoV-191,2, the origins of the virus remain unclear. Here, we report a novel bat-derived coronavirus, denoted RmYN02, identified from a metagenomics analysis of samples from 227 bats collected from Yunnan Province in China between May and October, 2019. RmYN02 shared 93.3% nucleotide identity with HCoV-19 at the scale of the complete virus genome and 97.2% identity in the 1ab gene in which it was the closest relative of HCoV-19. In contrast, RmYN02 showed low sequence identity (61.3%) to HCoV-19 in the receptor binding domain (RBD) and might not bind to angiotensin-converting enzyme 2 (ACE2). Critically, however, and in a similar manner to HCoV-19, RmYN02 was characterized by the insertion of multiple amino acids at the junction site of the S1 and S2 subunits of the Spike (S) protein. This provides strong evidence that such insertion events can occur in nature. Together, these data suggest that HCoV-19 originated from multiple naturally occurring recombination events among those viruses present in bats and other wildlife species.

Isolation, identification and phylogenetic analysis of a wild bird-derived H1N1 avian influenza virus in the northern Tianshan Mountain / 中华微生物学和免疫学杂志

Objective:To analyze the complete genome sequence and phylogenetic structure of a wild bird-derived H1N1 avian influenza virus (AIV) in the northern Tianshan Mountain.Methods:In November 2018, 320 samples of fresh wild bird feces were collected from several reservoirs in the middle part of northern Tianshan Mountain. Chicken embryo inoculation test, hemagglutination inhibition test and RT-PCR with PB1 universal primer were used to isolate and identify AIVs. Eight fragments of the viral genome were amplified with the universal primers of influenza A virus and the whole viral genome was sequenced. Pairwise sequence alignments and analysis of phylogenetic and molecular characteristics were performed by BLAST, Clustal W, MEGA7.0 and MegAlign software. Results:Influenza viruses were isolated and identified from six samples of wild bird feces with a positive rate of 1.88%. One of them was H1N1 AIV, named A/wild bird/Xinjiang/010/2018 (H1N1) (XJ-H1N1). The eight gene segments of XJ-H1N1 were all derived from AIVs isolated from wild ducks of Anseriformes. The surface genes of HA and NA were Eurasian lineages and derived from H1N1 isolated from Mongolian Anas platyrhynchos and H3N1 isolated from Bangladesh wild duck, respectively. The six internal genes were derived from H6N8 isolated from Anas strepera in Siberia, H7N3 isolated from Anas clypeata and teal in Egypt, and H7N5 isolated from wild birds such as Anas platyrhynchos in the Netherlands. The HA cleavage site of XJ-H1N1 contained only one basic amino acid, suggesting that it was a low pathogenic AIV. Amino acids at positions 190 and 225 of HA receptor binding sites were E and G (H3 count), which could bind both α2, 3 galactoside sialic acid (SAα2, 3Gal) and SAα2, 6Gal receptors. T200A and E227A mutations in HA amino acid sequences and P42S mutation in NS1 amino acid sequences could all enhance the replication ability and pathogenicity of the virus in mammalian cells. Conclusions:A low pathogenic H1N1 AIV, XJ-H1N1, was isolated from wild birds in the northern Tianshan Mountain, resulting from multiple reassortments of AIVs carried by migrating wild ducks. The replication capacity and pathogenicity of XJ-H1N1 in mammalian cells might be enhanced. Moreover, the virus could bind both SA 2-3gal and SA 2-6gal receptors.

Genetic evolution of HA and NA genes of H9N2 influenza viruses isolated in regions of Hunan Province, China, in 2015 / 生物工程学报

The high prevalence of influenza A virus is identified in Hunan Province because of the high density of poultry farms. To survey the variations of H9N2 subtype avian influenza virus in Hunan province, we analyzed HA and NA genes of 10 virus strains isolated from different areas of Hunan Province. All these strains belong to the Eurasian lineage, Y280-like sub-lineage. The cleavage sites in their HA genes were all RSSR↓GLT, corresponding to the feature of low pathogenic AIV. All strains had an L (Leu) at the site 234 in the HA genes, indicating the ability of binding with the SAα-2,6 receptor. NA gene stalk deletions at aa 63-65 were also detected from all the isolates, indicating a possibility of increased virus replication in mammals. Our findings suggest that more attention should be paid to the surveillance of H9N2 influenza virus and its direction of reassortment.

A novel carbon nanoparticle probe-based ultrasensitive lateral flow assay for rapid detection of Ebola virus / 生物工程学报

Ebola virus (EBOV) is an extremely contagious pathogen first discovered in Africa associated with severe hemorrhagic disease in humans and nonhuman primates, which has resulted in at least 28 500 suspected cases and 11 300 confirmed deaths in 2014-2016 Ebola epidemic in West Africa. Rapid and sensitive detection of EBOV is the key to increasing the probability of survival and reducing infection rates in pandemic regions. Here, we report an ultrasensitive and instrument-free EBOV detection assay based on colloidal carbon immunochromatography. Carbon nanoparticle-labeled rabbit anti-EBOV-VP40 IgG were concentrated in the conjugate pad, monoclonal antibody (McAb, 4B7F9) against EBOV-VP40 and goat anti-rabbit IgG were immobilized on the nitrocellulose membrane with 2 μL/cm at a concentration of 1 mg/mL as test and control lines, respectively. Then the sample application pad, conjugate release pad, nitrocellulose membrane and absorbent pad were assembled into a lateral flow test strip. The test strip shows strong specificity against related viruses that share similar clinical symptoms and geographic range with EBOV, including marburg virus, influenza virus, yellow fever virus and dengue virus. In addition, 1 500 negative serums were tested with false-positive rate of 1.3‰ which significantly lower than that of ReEBOV™ colloidal gold test kit recommended by World Health Organization (WHO). The sensitivity of this strip was analyzed using inactivated EBOV with detection limit of 100 ng/mL (10⁶ copies/mL) which clearly higher than that of ReEBOV™ dipstick (10⁸ copies/mL). Furthermore, the strip showed excellent thermal stability characteristics in room temperature and could be as a point-of-care (POC), ultra-sensitive and specific promising candidate for EBOV serological screening in rural Africa or entry/exit ports.